Helicobacter infections of human gastric epithelium cause gastritis, are a major factor in the development of peptic ulcers and gastric lymphoma, and may be a risk factor for the development of gastric cancer [1-3]. The most frequent infection agent is Helicobacter pylori, followed at a much lower frequency by Helicobacter heilmanii. H. pylori is a slender S-shaped gram negative microorganism, which is routinely recovered from gastric biopsies of adults and children with histologic evidence of gastritis or peptic ulceration. Evidence for a causal relationship between H. pylori and gastroduodenal disease comes from studies in human volunteers, patients with ulcers and gastric cancer, gnotobiotic pigs, and germ-free rodents. Regarding etiology, Koch's postulates were satisfied by creating histologically confirmed gastritis in previously uninfected individuals following consumption of viable microorganisms [4-11], and by treatment to eradicate H. pylori, with resolution of the gastritis and, in patients with peptic ulcer disease, a decrease in the recurrence rate [12].
In spite of in vitro susceptibility to many antimicrobial agents, in vivo eradication of established H. pylori infections with antimicrobial agents is often difficult to achieve [13]. The microorganism is found within the mucous coat overlying the gastric epithelium and in gastric pits. These are locations which do not appear to allow for adequate antimicrobial levels to be achieved even when antibiotics are given orally at high doses. At the present time, most authorities recommend a "triple therapy", namely a bismuth salt in combination with drugs such as tetracycline and metronidazole for 2-4 weeks. However, the effectiveness of this or other chemotherapeutic regimens remains suboptimal. Furthermore, this treatment may produce serious adverse drug reactions.
At the present time little is known regarding the role of the mucosal immune systems in the stomach. The distribution of immunoglobulin (Ig) producing cells in the normal gastric antrum indicates that IgA plasma cells make up 80% of the total plasma cell population. In addition, the number of plasma IgA cells present in the gastric antrum is comparable to other mucous membranes [14, 15]. A number of studies in humans [16] and in animal models [8,10] have demonstrated specific IgG and IgA responses in serum and in gastric secretions in response to Helicobacter infection. However, the observation that H. pylori infection persists as a chronic infection for years, despite inducing a local and systemic immune response, is not encouraging the development of immunization strategies.
Lee et al have reported the ability to infect germ-free rodents with Helicobacter felis, a bacterium closely related to H. pylori, and reproducibly document histologic gastritis [9, 10]. Since then, this bacterium-host pairing has been accepted as a good model to study Helicobacter-mediated gastritis and its initiating factors [17]. Czinn et al have shown that repetitive oral immunization with a crude lysate of H. pylori plus cholera toxin adjuvant induces a vigorous gastrointestinal IgA anti-H. pylori response in mice and ferrets [13]. In addition, Chen et al and Czinn et al have recently reported that oral immunization with a crude lysate of H. felis induced protection against H. felis infection in mice [21, 22]. The exact nature of the antigen(s) responsible for the induction of this protection, however, had not been determined, and no information suggested that the protective antigen(s) of H. felis that induced protection against this pathogen would induce a cross-reactive protection extending to another Helicobacter species.
We have demonstrated for the first time that H. pylori and H.felis share antigenic determinants by obtaining monoclonal antibodies after oral immunization of mice with either H. pylori or H. felis sonicates and showing that some of these antibodies, directed against H. pylori, would crossreact with H. felis and vice versa [24;25]. The basis for these cross-reactivities were unknown.
Based on the homology existing between the different known urease amino acid sequences, it has been proposed that urease could be used as a vaccine against H. pylori [26]. Nevertheless, despite the homology among the different ureases sequences, cross-reactivity is not the rule. Guo and Liu have shown years ago that ureases of Proteus mirabilis, Proteus vulgaris and Providencia rettgeri show cross-reactivity to each other, while ureases of jack bean and Morganella morganii are immunologically distinct from the three former ureases [23]. Even if an antigenic cross-reactivity of H. pylori urease with other Helicobacter ureases was a reasonable postulate, no data existed demonstrating that this was really the case until we showed that some H.felis monoclonal antibodies crossreacted with H. pylori urease [25]. J. Pappo has further demonstrated that mice which have been infected by H. felis produce antibodies which crossreact with H. pylori urease but not jack bean urease (J. Pappo, unpublished data, 1993).
The use of H. pylori urease, or of related ureases, as a vaccine against H. pylori infection has previously been proposed by A. Labigne in EPO 367,644 [28]. However, that application contains no evidence of vaccination of any mammal against any Helicobacter infection with urease.
Moreover, while sequence homology with other bacterial ureases might support the use of urease as a vaccine candidate against H. pylori infection, the current knowledge of human H. pylori infection would certainly not. First, despite the fact that infected individuals often mount a strong antibody response to urease, the anti-urease immune response does not result in clearance or control of the infection. Secondly, H. pylori is able to transport urease out of the cell and to shed it from its surface [19, 20]. Thus, urease may not represent an appropriate target for the development of a protective mucosal immune response. Indeed, mucosal immune protection is thought to be mainly mediated by secretory IgA, the agglutinating activity of which would be impaired when the recognized antigen can be shed by the target pathogen and thereby serve as a decoy for the protective antibody. Thirdly, urease appears to be toxic for epithelial cells in culture, and has been suspected to play a role in mucous degradation and in peptic ulceration in vivo. Thus, its use as antigen may be toxic.
Nevertheless, we reasoned that this antigen could be a potentially efficient vaccine if:
first, we would deliver it orally at a sufficiently high dose to elicit a stronger immune response than the naturally occuring one PA1 second, the amount of antibodies produced would be high enough to bind all the urease, shed or not shed PA1 third, we would use subunits of urease or a molecular species that was non toxic.
In summary, there remains a need for effective treatment and prevention of H. pylori-induced gastric infection in humans. Recent data suggested the possibility to generate a vaccine against this infection, but have not provided a clear identification of defined antigen(s), common to all strains of H. pylori, that could be incorporated into a safe and effective vaccine.
In this invention, we have identified the urease antigen of H pylori as a candidate vaccine and demonstrated its efficacy in an animal model. These results were unexpected in the light of the natural history Helicobacter infections.